Agreement between two real-time commercial PCR kits and an in-house real-time PCR for diagnosis of mucormycosis

ABSTRACT Mucormycosis is a severe and emerging invasive fungal infection associated with high mortality rates. Early diagnosis is crucial for initiating specific antifungal treatment, with molecular tools currently representing the most efficient diagnostic approach. Presently, a standardized in-house real-time PCR method is widely employed for diagnosing mucormycosis. Our study aimed to evaluate the agreement for the Mucorales DNA detection between two commercial real-time PCR assays—the Fungiplex Mucorales Real-Time PCR Kit and the MycoGENIE Aspergillus-Mucorales spp. Real-Time PCR Kit—in comparison with the in-house PCR. We retrospectively analyzed 58 samples previously identified as positive for Mucorales using the in-house PCR. These samples, obtained from 22 patients with proven or probable mucormycosis, were tested with both commercial kits. Additionally, samples from 40 patients without mucormycosis served as negative controls. Our findings revealed that the MycoGENIE Kit demonstrated superior performance in detecting Mucorales DNA in samples identified as positive by the in-house PCR. Notably, we observed minimal variability in cycle threshold (CT) values when comparing the results of the MycoGENIE Kit with those of the in-house PCR, with an average difference of 1.8 cycles. In contrast, the Fungiplex Kit exhibited a larger discrepancy in CT values compared to the in-house PCR, with an average difference of 4.1 cycles. The MycoGENIE Kit exhibited very good agreement (kappa of 0.82) with the in-house PCR for detecting Mucorales DNA across various sample types. These findings are important for the choice of kits that could be used to diagnose mucormycosis in clinical microbiology laboratories. IMPORTANCE Early diagnosis of mucormycosis is crucial for initiating effective treatment. The detection of Mucorales DNA by PCR in serum has revolutionized the diagnosis of this infection. However, the use of in-house methods can be time consuming. The availability of a commercial kit eliminates the need for in-house assay development, reducing laboratory workload and ensuring consistent performance across different healthcare settings. Currently, there are several commercial assays available, but many have limited evaluation. In this study, we compared two commercial kits and found that the MycoGENIE Kit offers a promising alternative to the in-house method.

of the diagnosis of the mucormycosis in order to initiate the specific treatment with liposomal amphotericin B and surgery (7,8).While mycological work-up (includ ing direct examination and culture) and histology remain the gold-standard diagnos tic methods according to the recommendations of the EORTC/MSGERC (European Organization for the Research and Treatment of Cancer/Mycoses Study Group Education and Research Consortium), their results may be delayed (9).Moreover, biopsies are not always available depending on the site of infection and the clinical condition of the patient (10).Recently, in-house real-time PCR assays have emerged as promising alternatives, demonstrating reliability in early detection (11).Notably, PCR in serum can be positive several days before the conventional diagnosis (12), making it a valuable screening tool for at-risk patients suspected of mucormycosis.Moreover, PCR has prognostic value and can be used to monitor the antifungal treatment efficacy (11).
Currently, three commercial kits are available for detecting Mucorales DNA in serum, respiratory samples, and biopsies (MucorGenius by PathoNostics, MycoGENIE Mucorales by Ademtech, and Fungiplex Mucorales by Bruker).MucorGenius is the kit that has been the first to be evaluated (13)(14)(15)(16)(17)(18)(19).Furthermore, the MycoGENIE Kit was recently prospectively evaluated as a molecular workflow for the diagnosis of both mucormycosis and aspergillosis in sera from high-risk patients (20).Currently, the in-house Mucorales real-time PCR developed by Millon et al. (18,21) has been standardized and evaluated and is adopted by numerous laboratories.Nevertheless, for routine laboratories, the availability of commercial kits is of importance as in-house PCR is time consuming and labor intensive.Our study aims to evaluate the agreement between the results obtained with the MycoGENIE Mucorales and Fungiplex Mucorales Kits and those of in-house PCR, within our center.
A total of 40 samples from 40 different patients without mucormycosis collected in 2022 were selected to serve as negative controls for the two Mucorales PCR kits.Among these 40 patients, 20 (20 samples) had a negative result with both in-house Mucorales PCR and specific Aspergillus fumigatus PCR, and 20 other patients (20 samples) had a negative result by Mucorales in-house PCR but had a positive result by specific Aspergillus fumigatus PCR assay.As aspergillosis is the most frequent filamentous fungal infection, we have included 20 samples that were positive for Aspergillus to ensure that Mucorales DNA detection was not falsely detected in these samples.The samples included 18 respiratory samples, 18 blood samples, 1 cerebrospinal fluid (CSF), and 3 other types of samples.

Positive control strains
Seven strains of Mucorales belonging to six species cultured on Sabouraud supplemen ted with antibiotics (chloramphenicol and gentamicin) incubated at +27°C were used as positive controls for Mucorales PCR: one Cunninghamella bertholletiae, one Rhizopus arrhizus, two Rhizomucor pusillus, one Mucor circinelloides, one Lichtheimia corymbifera, and one Mucor irregularis (formerly Rhizomucor variabilis).

Processing of samples and DNA
For biopsies and other purulent or viscous samples, a pretreatment was performed using a MagNA Lyser apparatus (Roche Diagnostics, Meylan, France).
One milliliter of serum, respiratory samples, or biopsies and 5.0 µL of internal control were extracted using an EMAG automatic device (bioMérieux, Marcy l'Etoile, France); 50 µL of DNA eluates was obtained for serum and 100 µL for respiratory samples and other types of samples.

Mucorales strains and DNA extraction
DNA extraction from Mucorales strains was performed manually using the MasterPure Yeast DNA Purification Kit (Lucigen, LGC, Biosearch Technologies, USA), according to manufacturer's instructions, and DNA pellet was resuspended in 100 µL of sterile distilled water.

Real-time Mucorales PCR assays
The in-house Mucorales real-time PCR assay, described and validated by Millon et al. (12), was performed in routine diagnostic at the Parasitology-Mycology Laboratory of Necker Children's Hospital, Paris .The PCR assay, targeting the 18S rDNA, included a simple PCR for the detection of Mucor-Rhizopus spp.and a multiplex PCR for the detection of the two genera Lichtheimia spp.and Rhizomucor spp.PCR reactions were performed on CFX96-Biorad apparatus.The CT cut-off value for in-house PCR is <40.Two real-time PCR commercial kits-Fungiplex Mucorales RUO Real-Time PCR Kit (Bruker, Bremen, Germany) and MycoGENIE Aspergillus-Mucorales spp.(Ademtech, Pessac, France)-were blinded and evaluated on the same samples that are used for in-house Mucorales PCR, by using DNA eluates kept frozen at −80°C.

Detection of DNA from positive control strains
Among the seven strains belonging to five genera/six species, in-house PCR detected DNA from all species except C. bertholletiae and provided accurate identification at the genus level for Lichtheimia and Rhizomucor, and at the group level for Mucor/Rhizopus.The MycoGENIE Kit detected DNA from all the six species, but the kit is not designed to provide identification at the genus/species level.Finally, the Bruker Kit detected DNA from all species except M. irregularis (formerly Rh. variabilis) and gave an accurate clustering into the three groups of Mucorales identified by the three different fluorochromes.

Performances of the two commercial kits (Fungiplex and MycoGENIE) in comparison to in-house Mucorales PCR for DNA detection in clinical samples
A total of 53 samples (47 positive samples by in-house Mucorales PCR and 6 negative samples) were used to test the two kits, Fungiplex and MycoGENIE, in parallel (Fig. 1, step 1).Among the 47 positive samples, 39 (83%) were positive by the MycoGENIE (8 false positive), while only 24 (51%) were positive by the Fungiplex Kit (23 false positive) (Table 2).All the six negative samples were also negative with both commercial kits.
We compared the CT values obtained for each of the 53 samples using the three methods (Fig. 2).The 39 positive PCR results with MycoGENIE Kit showed CTs close to that of the in-house PCR with an average CT difference of 1.6 cycles (IQR, 0.1-5).For the 24 positive PCR results with Fungiplex, the difference in CT with that of the in-house PCR was larger, with an average of 4.1 cycles (IQR, 0.6-8.4).
The eight false negative results obtained by the MycoGENIE had high CTs of ≥35 by in-house PCR.For the Fungiplex Kit, only 7 out of the 23 negative PCR had high CTs of ≥35 by in-house PCR.
The agreement between the two kits has been calculated by the kappa statistic.The kappa value was 0.18, corresponding to a poor agreement.

Agreement between the MycoGENIE Kit and the in-house PCR
Agreement between the MycoGENIE Kit and the in-house PCR was evaluated using the data from all the 98 samples (Fig. 1, step 2).Among the 58 positive samples with the in-house Mucorales PCR, 49 samples were tested positive with the MycoGENIE Kit.Among the 40 negative control samples, 20 were positive for the detection of Aspergillus fumigatus DNA (Table S1), but all the 40 samples were detected negative for Mucorale DNA with the MycoGENIE Kit.The agreement between the MycoGENIE and in-house PCR was very good with a kappa value of 0.82.
Comparison of the CT values obtained with the MycoGENIE Kit and in-house PCR showed very low variability (Fig. 4).The average CT difference was 1.8 cycles (IQR, 0.1-5).Thus, the coefficient of variation is on average 3%, ranging from 0.1% to 11.2%.However, it should be noted that all six samples that tested positive by in-house PCR but negative by MycoGENIE Kit had CT values of ≥35 in the in-house PCR.

DISCUSSION
Mucorales infections are distinguished by a notable release of Mucorales DNA, detecta ble in tissues and present in high amount in the bloodstream.DNA detection serves as a robust diagnostic marker for mucormycosis.Extensive research by L. Millon's team has underscored the diagnostic and prognostic significance of DNA detection across various clinical forms-be it rhinocerebral, cutaneous, pulmonary, or disseminatedand irrespective of the genus involved (11,12,18).A recent clinical study led by this group advocates for the integration of Mucorales PCR as a fundamental diagnostic tool for these deadly infections (11).This recommendation stems from the utilization of a meticulously standardized and assessed in-house PCR assay, albeit not commercially available.
Concurrently, several manufacturers have developed kits for this purpose, yet only a handful have undergone comparative evaluation against the in-house PCR using identical clinical samples.Our study addresses this gap by assessing two CE-stamped kits against the in-house PCR on a substantial number of positive samples.The objective is to ascertain their potential as alternatives to the in-house PCR.Our findings reveal that one of the kits, namely the Fungiplex Kit, exhibits inadequate performance to supplant the in-house PCR.On the other hand, the second kit, MycoGENIE, demonstrates superior performance, although it still falls short of the accuracy achieved by the in-house PCR.
The diagnostic value of the MycoGENIE Kit has been previously evaluated in BAL (23) and serum (20), but in none of these studies, the analytic performances of this kit have been compared to the performance of the in-house PCR.It has to be noted that there is no published study on the evaluation of the Fungiplex Kit.Evaluation of the commercial kits is of prime importance to allow their integration in the workflow of the routine diagnosis in clinical microbiology laboratories.The development of the molecular biological techniques is crucial to improve the early diagnosis of the mucormycosis infection to start specific treatment (antifungal and surgical treatment) without delay.It is well known that the delay in specific treatment is associated with a high mortality rate (7).It has to be noted that, up to now, guidelines for the diagnosis of mucormycosis only moderately recommend the use of PCR methods (9) due to the lack of standardization of these methods.This highlights the need for evaluation of standardized commercial kits.On the other hand, while mycological culture and histological examination are very specific, their sensitivity remains low for biological diagnosis.The difficulty of the mycological culture and the histological examination is related to the accessibility of the lesions and the processing time of the samples.On contrary, blood samples are easy to obtain and can be used for molecular testing in both diagnosis and screening strategies (11).
Our study evaluated the performance of two commercial kits for the detection of Mucorales DNA and showed the better performance of the MycoGENIE Kit in comparison to the Fungiplex Kit.Indeed, up to now, no study has compared the efficiency of these two kits.Although, according to the supplier's recommendation, a lower volume of DNA extract was used for the reaction (10 µL for the MycoGENIE Kit and 5 µL for the Fungiplex Kit), this could not fully explain the differences in performances between the two kits.One of the drawbacks on the MycoGENIE is the inability to identify the genus and species of Mucorales because all the PCR targets were detected on the same channel.On contrary, the Fungiplex Kit allowed to identify a set of Mucorales species as three different channels is used to run the assay.Moreover, it should be noted that some species are not detected by Fungiplex Kit in comparison with MycoGENIE such as M. irregularis (formerly Rh. variabilis).
There are some limitations of the present study.The most important is the small sample size and the variety of samples included.However, this study encompassed the most prevalent samples utilized for the real-life diagnosis of mucormycosis.Notably, 54% of the samples were derived from blood, while 20% originated from the respiratory tract.Furthermore, the study incorporated invaluable invasive samples such as CSF, cutaneous biopsies, and sinus biopsies, which corresponded to the specific localization of the infection.Here, our study provides information on agreement between MycoGENIE and in-house PCR for DNA detection, but adequate evaluation of diagnostic accuracy needs to be tested in prospective real-world studies.
In conclusion, our study showed that the MycoGENIE Kit provides good performance but remains inferior to in-house PCR.Nevertheless, the MycoGENIE Kit does not provide any information regarding the genus responsible for infection.The current availability of reliable commercialized kits and their intrinsic performance validation opens the way to standardize molecular diagnosis of mucormycosis in routine clinical microbiology laboratories.

FIG 1
FIG 1 Flow chart of the study.In a first step, 53 samples (47 positive and 6 negative) were used to test the performances of DNA detection by the two commercial kits (Fungiplex and MycoGENIE) in comparison to the in-house PCR.In a second step, all the 98 samples (58 positive and 40 negative) were used for the evaluation of the agreement for DNA detection between the sensitivity and specificity of the MycoGENIE Kit and the in-house PCR.

FIG 2
FIG 2 Comparative cycle threshold values obtained by the two PCR kits (Fungiplex, orange dots; MycoGENIE, blue dots) and the in-house Mucorales PCR (gray dots).The 47 clinical samples containing Mucorales DNA detected by the in-house PCR are sorted in ascending order of the CT value.

FIG 3
FIG 3 DNA detection by two PCR Mucorales kits compared to the result of the in-house PCR according to the genera of Mucorales.Each bar represents the number of positive samples obtained by the in-house PCR assay (gray bar), the Fungiplex Kit (orange bars), and the MycoGENIE Kit (blue bars).

FIG 4
FIG 4 Comparative cycle threshold values obtained by the MycoGENIE PCR kit (blue dots) and the in-house PCR (gray dots).The 58 clinical samples containing Mucorales DNA detected by the in-house PCR are sorted in ascending order of the CT value.

TABLE 1
Characteristics of patients and samples a a HSCT, hematopoietic stem cell transplant; GVH, graft versus host disease; AML, acute myeloid leukemia; KID syndrome, keratitis ichthyosis deafness syndrome; ALL, acute lymphoid leukemia; ENT, ear nose throat; BAL, bronchoalveolar lavage; CGD, chronic granulomatous disease; NA, not available.

TABLE 2
PCR results obtained by the two Mucorales kits compared to in-house PCR